Sustained Release of Hydrogen Sulfide from Di(t-butanol)dithiophosphate Phenethylamine Salt Encapsulated into Poly(lactic acid) Microparticles to Enhance the Growth of Radish Plants

The slow release of hydrogen sulfide has been shown to be beneficial to plants by protecting them from environmental stressors, increasing germination, and extending the lifetime of harvested fruits. A major challenge in this field is controlling the amount and location of release of hydrogen sulfide so that it is available for use by plants at optimal amounts. This article reports a dual method to release hydrogen sulfide near the roots of plants by controlling its release using the hydrolysis of a dithiophosphate and the degradation of poly(lactic acid) [PLA]. Di(t-butanol)dithiophosphate phenylethylamine (tBDPA) was dissolved in a solution of PLA, and the solvent was allowed to evaporate. The resulting solid was crushed in a blender and separated into microparticles with two different size distributions of 250–500 or 500–2000 μm. The microparticles were characterized by powder X-ray diffraction to measure the presence of microcrystals of tBDPA within PLA, and images obtained using scanning electron microscopy with energy dispersive X-ray analysis confirmed the presence of these crystals. Microparticles of tBDPA loaded within PLA were characterized for their release of phosphorus and hydrogen sulfide, which both showed a burst release within 3 days, followed by a steady release. Radish plants grown with microparticles of PLA loaded with tBDPA had up to a 141% increase in harvest yield compared to plants grown in the presence of free tBDPA not loaded into PLA, PLA microparticles without tBDPA, and control plants grown without PLA or tBDPA. These experiments showed that loading hydrogen sulfide-releasing chemicals into PLA is a promising method to improve the effect of hydrogen sulfide on plants.


■ INTRODUCTION
Hydrogen sulfide (H 2 S) is a key gasotransmitter in plants that affects numerous enzymatic pathways within, and between cells, 1−10 and the addition of exogenous H 2 S often has a strong positive effect at surprisingly low doses on maize, soybean, wheat, cucumber, peas, tomatoes, broccoli, lettuce, sugar beets, strawberries, radishes, and kiwi plants. 6,11−24 H 2 S is produced in cells and found at nanomolar concentrations, and recent work has shown that applying exogenous H 2 S to plants has dramatic effects, including increasing their overall size and mass, protection from high salt concentrations, protection from heat and drought stress, increased root growth, and prolonged shelf life of harvested crops. 7,21,22,25−35 For example, the addition of exogenous H 2 S to plants such as over a dozen different plants increases their ability to withstand salt stress. 33,35 Although the process by which exogenous H 2 S enables plants to survive salt stress is complex, it has been shown to increase the activity of antioxidant enzymes, affect the levels of Na + in cells, and regulate several signaling proteins. 36−39 The importance of exogenous H 2 S in agriculture has been established, and many current studies investigate the fundamental biochemistry of H 2 S within plants and how to apply H 2 S in a safe, dependable manner. 6 Most of the early work in this field used micro to millimolar concentrations of aqueous H 2 S delivered once or twice daily. Although this work showed that exogenous H 2 S could improve the growth and survival of plants, most of the H 2 S evaporated due to its low boiling point of −60°C, which led to an intense smell of H 2 S around the plants and an unknown amount of H 2 S that was adsorbed by the plants. To address these challenges, scientists used chemicals such as GYY-4137, which slowly released H 2 S by hydrolysis (Figure 1a). 40,41 A low, one-time dose of milligrams of GYY-4137 per seed was required to have these strong, positive effects on plants. 22 In 2019, our group demonstrated that dialkyldithiophosphates release H 2 S in water at controlled rates were stable for months in the solid state and had potential applications in agriculture. Maize grown for 4.5 weeks after exposure to 1−200 mg per seed of dibutyldithiophosphate had an increased weight of plants by up to 39% compared to control plants not exposed to dibutyldithiophosphate. 6 Although the application of H 2 S is an exciting new frontier in agriculture, it is challenging to deliver at known doses over specified periods of time, which are two important parameters for fundamental studies of the effect of H 2 S on plants and for eventual commercial applications. 42 Chemicals such as GYY-4137 address some of these challenges, but the rate of release of H 2 S is not easily varied from chemicals with its general structure, and it hydrolyzes to release unsafe chemicals such as morpholine that have unknown effects on plants. 43,44 Furthermore, small chemicals such as GYY-4137 and dithiophosphates are water-soluble and can wash away from the roots of plants. What is needed in this field is a method to apply chemicals that slowly release H 2 S over well-defined time periods, release biocompatible and safe chemicals in addition to H 2 S, and will not diffuse away from the roots of plants. In this paper, we report the encapsulation of a dithiophosphate into microparticles of poly(lactic acid) [PLA] and their effect on radish plants (Figure 1b). The PLA microparticles are stationary in soil and degrade at well-understood rates. 45−49 Dithiophosphates or other chemicals encapsulated within PLA microparticles are released at controlled rates near where the microparticles were applied within soil rather than be washed away with water. Furthermore, the degradation of PLA provides a method to provide a slow, controlled release of dithiophosphates and H 2 S near the roots of plants.
PLA was used for preparing the microparticles because it merges several interesting properties that make it an ideal candidate for our studies. PLA is a stable, odorless, and inexpensive polymer that is produced on an industrial scale. 50−52 It naturally degrades through hydrolysis to release lactic acid and other chemicals. 46−55 It is a key polymer in medicine to encapsulate and provide the slow release of pharmaceutical drugs. It has been approved for use in medicine, and it is used to fabricate numerous commercial products such as straws, cups, lids, landscape fabric, mulch film, and cutlery. 56−66 In our prior work, we synthesized dithiophosphates from reactions between alcohols or thiols with P 4 S 10 and demonstrated that these chemicals hydrolyze in water at rates that varied by a factor of over 10 4 . 44 Dithiophosphates hydrolyzed in water to release H 2 S as well as phosphoric acid and the alcohols or thiols used in their synthesis. By selection of biocompatible alcohols or thiols, the hydrolysis products of dithiophosphates were safe for the environment and unlikely to have a strong effect on plants which allows the response of plants to H 2 S to be measured. In addition, dithiophosphate salts are solids with long shelf lives (stable over 6 months). 6 In this paper, we describe the encapsulation of a dithiophosphate into microparticles of PLA. Encapsulation of a dithiophosphate within PLA addresses the critical problem of providing a sustained release of H 2 S near the roots of a plant because the PLA particles will remain in close proximity to where they were originally added in the soil. The microparticles have a slow, sustained release of H 2 S as the PLA degrades. In this article, we report preparation of different microparticles with different sizes and compositions, release of a dithiophosphate salt from the microparticles, and H 2 S release from microparticles in buffered water. To demonstrate the potential applications of H 2 S-releasing microparticles in agriculture, we also describe their effects on the growth of radish plants after exposure to milligram-loading of microparticles. This H 2 S delivery system provides a solution for a sustained, localized delivery of H 2 S over weeks.

■ EXPERIMENTAL SECTION
Materials and Methods. PLA [MW 150 000 g/mol] was purchased from Indeo. All other chemicals were obtained from Sigma-Aldrich at their highest purity and used as received. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance-300 at 300 MHz and a Bruker DRX-400 at 400 MHz. Powder X-ray diffractometry (pXRD) was performed using a Siemens Model D5000 X-ray diffractometer (Bruker AXS Inc., WI). Scanning electron microscopy (SEM) and SEM-energy dispersive X-ray (SEM-EDX) microscopy of microparticles were performed on an S-2700 scanning electron microscopy (Hitachi, Japan). Differential scanning calorimetry (DSC) of microparticles was completed on a DSC TA Q100 instrument.
The radish seeds were purchased from Earl May Seed and Nursery. Potting mixes were obtained from Beautiful Land Products in West Branch, IA. Potting mix #4 was a peat/bark-based general-purpose growing mix and was used in these experiments. Pots were 2.5″ TEKU VCC 15 US 0600 (1.48 L) purchased from Hummert International.
Synthesis of Di(tert-butanol)dithiophosphate Phenethylamine Salt (tBDPA). tert-Butanol (0.71g, 9.54 mmol) was added slowly over 2 min to a mixture of P 2 S 5 (0.52 g, 2.34 mmol) and THF (15 mL) under nitrogen. The contents were stirred at 45 ± 0.5°C under a nitrogen atmosphere for 5 h using a thermostat-controlled hot plate/stirrer, and the contents were purged with pure dry nitrogen set at a pressure of 20 psi and flow rate of 10 mL/min. The excess nitrogen gas was vented via a bubbler. The contents were cooled in an ice bath, and phenethylamine (0.66 mL, 5.22 mmol) was added slowly over 2 min. The pure product was obtained after washing with hot toluene and dried under reduced pressure to give a white solid (85% yield). 1  Microparticle Fabrication Using a Modified Solvent Evaporation Technique. Microparticles with 16.7% loaded tBDPA in PLA (w/w) were prepared using a modified solvent evaporation technique. First, the PLA polymer (20.0 g) was dissolved in DCM (400 mL), and then methanol was added (8% v/v). tBDPA (4.02 g) was then added and stirred until dithiophosphate and PLA were both completely dissolved. After obtaining a clear solution, the solvent was removed under reduced pressure. The final solid was colored white. Microparticles were obtained by agitation within an Oster Classic Series blender. Next, the microparticles were separated into different sizes using sieves with different aperture sizes. Microparticles with sizes between 500−2000 and 250−500 μm were collected separately.
Microparticle Fabrication with Phenylethylamine Hydrochloride Salt. Microparticles with 16.7% phenethylamine hydrochloride salt in PLA (w/w) were prepared using a modified solvent evaporation technique. The PLA polymer (20.01 g) was dissolved in DCM (400 mL). Phenethylamine hydrochloride (4.0 g) was added and stirred until the amine was completely dissolved. The solvent was removed under reduced pressure to yield with a white sold. Microparticles were obtained by agitation within an Oster Classic Series blender. Next, the microparticles were separated into different sizes using sieves with different aperture sizes. Microparticles with sizes between 500−2000 and 250−500 μm were collected separately.

Scanning Electron Microscopy (SEM) and SEM-Energy Dispersive X-Ray (SEM-EDX) Microscopy of Microparticles.
The shape and surface characteristics of the microparticles were investigated using SEM. The particles were placed on an aluminum specimen stub using adhesive carbon tape. The mount was then coated by ion sputtering with conductive gold set at 10 mA for 2.5 min and examined using SEM operated at a 2 kV accelerating voltage.
Powder X-Ray Diffractometry (pXRD). The samples were exposed to Cu Kα X-rays with a voltage of 40 kV and a current of 50 mA. The scanning angle was recorded from 10 to 80°at 25°C at a step size of 0.020°. The scanning was obtained in a continuous scan mode.
Differential Scanning Calorimetry (DSC) of Microparticles. The thermal behavior of microparticles was investigated by DSC. Samples were sealed in standard aluminum sample pans, and an empty sealed aluminum pan was used as a reference. Samples were purged with pure dry nitrogen set at a pressure of 20 psi and flow rate of 20 mL/min. DSC thermograms were obtained for both PLA polymer and PLA microparticles loaded with tBDPA by heating the samples from 30 to 180°C with a heating rate of 10°C/min.

Release of tBDPA from Microparticles and Determination of Percentage Mass Loss from Microparticles at pH 7.2.
Microparticles (0.300 g) with dimensions of 500−2000 μm and loaded with 16.7% tBDPA were placed in the 25 mL scintillation vials and suspended with 20 mL BIS−TRIS buffer solution (0.01 M, pH 7.2). The vials were placed on an orbital shaker at 100 rpm at room temperature and covered with an aluminum foil. Aliquots (1 mL) were taken from each vial separately at the specific time points and treated with a known concentration of methylenediphosphoric acid in D 2 O (0.016 M), which was used as an internal standard. The remainder of the vial was preserved to determine the mass loss percentage. The concentrations of tBDPA released into the solution were determined using 31 P NMR spectroscopy. Each experiment was performed in triplicate.
To determine the mass loss, the remainders of the solution within the vials were used. The solution above the microparticles was pipetted out carefully, and the microparticles were dried under reduced pressure until a constant mass was obtained. This mass was compared to the initial mass of the microparticles (0.3 g). Each experiment was performed in triplicate unless stated otherwise.
Investigation of H 2 S Release from Microparticles through a Modified Methylene Blue Reaction. To a 50 mL centrifuge tube, 0.30 g of 16.7% loaded microparticles and 10 mL of phosphate buffer solution (0.01 M, pH = 6.7) were added. A methylene blue solution (1.0 mL) was prepared in a disposable 10 mL test tube and placed inside the centrifuge tube and capped tightly. The methylene blue solution contained 0.5 mL of 0.1 M PBS (pH 6.7), 0.2 mL of 30 mM FeCl 3 in 1.2 M HCl, 0.2 mL of 20 mM N,N-dimethyl-p-phenylene diamine in 6.7 M HCl, and 0.1 mL of 1% (w/v) Zn(OAc) 2 . The centrifuge tube was sealed and then covered with aluminum foil to avoid oxidation of H 2 S by sunlight. At selected times, the test tube was removed from the centrifuge tube, and absorbance values at 670 nm were measured. Each experiment was performed in triplicate. H 2 S release from the particles was measured by comparing to the standard curve prepared with NaHS (10, 20, 30, 40, 50, 60, and 70 μm). The same procedure was followed for 9.1% loaded tBDPA microparticles and 16.7% loaded phenethylamine hydrochloride microparticles.
Growth of Radish Plants. Radish seeds were planted on October 08, 2021, in 2.5″ TEKU pots. The pots were packed finger-tight with potting mix #4 from Beautiful Land Products. The radish seeds were planted ∼1.5 in. deep. For the growth studies, 500−2000 μm-sized particles with 16.7% loaded tBDPA were used, and 30 seeds were planted at each loading (1, 3, 10, 30, 100, 300, and 600 mg). An additional 30 radish seeds were planted at each loading with PLA microparticles with dimensions of 500−2000 μm, and an additional 30 seeds were planted at each loading with free tBDPA. The amount of free tBDPA at each loading was calculated based on the amount of tBDPA within the loaded microparticles. At each loading along the xaxis, the amount of tBDPA was constant between experiments with microparticles loaded with tBDPA and experiments with free tBDPA. Radish plants were grown with 30 mg of 16.7% loaded phenethylamine hydrochloride microparticles as a control experiment. After the seeds were added to the soil, chemicals were added on the top of the seed and covered with the soil. Next, the plants were placed inside a greenhouse and watered daily. Radishes were harvested on November 12, 2021. After cutting off the shoot and brushing off the potting mix, roots were weighed on a laboratory balance.
Statistical Analysis. Statistical analysis was performed using IBM SPSS Statistics 25. A nonparametric Kruskal−Wallis test was performed to determine the significance. Data represented are means ± standard errors, with two asterisks (**) indicating a 99% confidence interval.

■ RESULTS AND DISCUSSION
Selection of a Dithiophosphate Salt. The goal of this project was to place PLA microparticles loaded with dithiophosphate in soil to slowly release hydrogen sulfide near roots to improve the growth of plants. To accomplish this goal, dithiophosphates released from the PLA particles must rapidly hydrolyze to release H 2 S before the dithiophosphates diffuse away. The first step was to select a dithiophosphate that had a rapid rate of hydrolysis, and that could be encapsulated within PLA. In a prior publication in 2021, the rates of hydrolysis of dithiophosphates synthesized from alcohols (primary, secondary, and tertiary), thiols, dithiols, and diols were investigated, and the two chemicals in Figure 2a had rates of hydrolysis that were >50× faster than the others. 44 The dithiophosphate synthesized from mercaptoethanol was not selected for this project because mercaptoethanol is toxic, and it rapidly cleaves disulfides that are present in proteins within plant cells. Dithiophosphates with tertiary alcohols were selected for this project due to the safety of t-butanol in the environment, the range of tertiary alcohols available, and the lack of odor of these alcohols. In prior work, it was shown that di(tbutanol)dithiophosphate potassium salt had a rapid rate of hydrolysis at room temperature in water buffered at a pH of 7.4 with a half-life of 5.7 days. Furthermore, the potassium salt was a solid and stable at room temperature for over 6 months. The complete hydrolysis of di(t-butanol)dithiophosphate released 2 equiv of t-butanol, 2 equiv of H 2 S, and phosphate salt (Figure 2b). Both phosphate (commonly used as fertilizer) and t-butanol are safe chemicals in the environment. 44 To fabricate dithiophosphates within the microparticles of PLA, it is desired to have both PLA and dithiophosphate soluble in the same solvent. The potassium salt of di(tbutanol)dithiophosphate was poorly soluble in methylene chloride, ethyl acetate, and acetonitrile, which are the solvents that dissolve PLA. To improve the solubility in organic solvents of dithiophosphate salts synthesized from tertiary alcohols, several dithiophosphates were synthesized, as shown in Figure 3. These chemicals were chosen because they are likely to be safe for the environment. 2,5-Dimethyl-2,5hexanediol is a safe alcohol commonly used in perfumes, pinacol is a starting material widely used in industry that has no significant concerns outlined in its safety data sheet, and trimethylbenzyl alcohol (commonly known as cherry propanol) is found in food such as carrots, fruits, citrus, and tomatoes. 67− 69 The reactions to synthesized dithiophosphates D, E, and F were completed at 45°C. The potassium and imidazolium salts were isolated to investigate their solubilities in methylene chloride, ethyl acetate, and acetonitrile. Unfortunately, both potassium and imidazolium salts of D and F were insoluble in these solvents. Moreover, hydrolysis of compound D in 90% H 2 O/D 2 O was investigated at 85°C and room temperature by 31 P NMR spectroscopy. After 30 days, only 18% hydrolyzed at 85°C and less than 3% hydrolyzed at room temperature. The chemical E was soluble in dichloromethane, but its rate of hydrolysis was also slow in 90% H 2 O/D 2 O at room temperature and 85°C, as measured by 31 P NMR spectros-  copy. Less than 3% of E hydrolyzed after 30 days at room temperature and only 20% hydrolyzed after 30 days at 85°C.
Di(t-butanol)dithiophosphate had the fastest rate of hydrolysis of the dithiophosphates studied, 44 so to improve its solubility in organic solvents, different bases were used to form its salt. The choice of bases was limited to chemicals that had a history of safety in the environment and that would be expected to have little effect on plants. Of the six amines investigated, as shown in Figure 4, only the dithiophosphate G was soluble in methylene chloride, ethyl acetate, or acetonitrile.
The chemical G was synthesized by reacting di(t-butanol)dithiophosphoric acid with pheneythylamine. In mammals, phenethylamine is produced from the amino acid L-phenylalanine by the enzyme aromatic L-amino acid decarboxylase via enzymatic decarboxylation. In addition to its presence in mammals, phenethylamine is found in many other organisms and foods�such as chocolate�after microbial fermentation. Salts of phenethylamine are sold as dietary supplements, and it is indigested orally to improve athletic performance, depression, weight loss, mood, and attention. This data suggests that use of pheneythylamine is not hazardous and will not pollute the environment when used in low to moderate amounts. 70−73 Synthesis and Characterization of PLA Microparticles Containing Di(t-butanol)dithiophosphate Phenethylamine Salt (tBDPA). PLA microparticles loaded with different amounts of tBDPA were prepared using a modified solvent evaporation method. The full details are described in the Experimental Section, but some of the key points are outlined here. PLA polymer (20.00 g) was dissolved in 400 mL of methylene chloride and 32 mL of methanol. The methanol was added to increase the solubility of tBDPA. Next, tBDPA (4.00 g) was added, and the solution was stirred until it was completely dissolved. The solvent was allowed to evaporate, and the PLA/dithiophosphate salt was obtained as a white solid. The solid was added to a blender and crushed to yield small particles that were separated into different-sized particles using sieves.
The composition of the loaded PLA microparticles was analyzed by powder X-ray diffraction (pXRD). The pXRD spectra of PLA, crystals of tBDPA, and PLA loaded with 16.7% tBDPA are shown in Figure 5. The pXRD spectrum of PLA was consistent with prior reports, and the pXRD spectrum of PLA loaded with 16.7% tBDPA showed that the PLA contained crystals of tBDPA. 74 Although PLA and dithiophos-phate salt were soluble in the methylene chloride/MeOH mixture, it is likely that some tBDPA precipitated as the solvent evaporated.
The structure and composition of the microparticles were further investigated by SEM and SEM-EDX mapping ( Figure  6). The PLA microparticles with sizes from 250 to 500 μm and loaded with 16.7% tBDPA were imaged by SEM (Figures 6c  and S5). These images showed that the particles had rough edges and irregular sizes as expected based on crushing loaded PLA within a blender and separation by sieves. The loaded PLA microparticles were imaged at high magnification before exposure to water (Figure 6a,b). The surfaces appeared rough, and an SEM-EDX map showed that sulfur was concentrated in selected areas. These areas are likely due to the crystals of tBDPA within the PLA matrix, which was consistent with the results from the pXRD spectrum that showed crystals of tBDPA within PLA. Moreover, exposure of loaded PLA to water for 30 days showed fractures in the surface of the PLA (Figure 6d). These fractures may have been voids created by the release of tBDPA crystals from the PLA.
DSC thermograms of PLA microparticles and PLA microparticles loaded with 16.7% tBDPA were obtained (Figure 7). The DSC trace of the PLA polymer exhibited an endothermic peak, T g , at 62°C, and this peak was observed at 56°C for PLA loaded with the dithiophosphate salt. An additional broad peak was observed at 124°C for PLA loaded with dithiophosphate salt that was assigned to the melting of the microcrystals of the salt. To confirm this assignment, the melting point of tBDPA was measured using a capillary melting point apparatus to be 130°C.
Release of tBDPA from Microparticles. The release of tBDPA from microparticles was investigated in water buffered at pH values of 6.0 and 7.2. Particles with two different size distributions were selected: 500−2000 μm-sized particles and 250−500 μm-sized particles. Numerous sets of microparticles were loaded into buffered water at loadings of 15 mg/mL. The vials were constantly agitated, and at defined time points, a vial was removed, the amount of phosphorous in the buffered water was measured using 31 P NMR spectroscopy, the microparticles were isolated and dried, and the dried microparticles were weighed. The experiments were repeated in triplicate, and the error bars for the measurements are shown in Figure 8.
The results in Figure 8 showed several important aspects of the degradation of the tBDPA-loaded microparticles. Microparticles loaded with 16.7% tBDPA had an initial rapid release of phosphate, and then a slower sustained release for both size distributions (Figure 8a). This initial rapid release was observed for other chemicals loaded into PLA in prior work. 75,76 After 30 days, the 500−2000 μm-sized particles released 61% of the tBDPA at a pH of 7.2 and 74% at a pH of 6.0, and the 250−500 μm-sized particles released 66% of tBDPA at a pH of 7.2 and 77% at a pH of 6.0. The weight of the PLA microparticles loaded with 16.7% tBDPA followed a similar trend with an initial burst loss of weight, followed by a slow, steady loss of weight (Figure 8c).
PLA microparticles loaded with 9.1% tBDPA had a similar rapid release of phosphate within 3 days, followed by a slower release (Figure 8b). After 30 days, the 500−2000 μm-sized particles released 37 and 44% of phosphate at pH values of 7.2 and 6.0, respectively. The 250−500 μm-sized particles released 43 and 51% of phosphate at pH values of 7.2 and 6.0, respectively. In both sets of microparticles, the 250−500 μm- The more rapid release at a pH of 6.0 compared to 7.2 can be understood by the acidcatalyzed hydrolysis of PLA, which facilitated the release of tBDPA. 53 Evaluation of H 2 S Release from PLA Microparticles Loaded with tBDPA. The release of H 2 S from PLA microparticles loaded with tBDPA was investigated using a modified methylene blue method. The methylene blue method traps H 2 S as ZnS, which is isolated as a solid. The ZnS is then reacted with HCl, FeCl 3 , and N,N-dimethyl-p-phenylene diamine to yield methylene blue. The adsorption of methylene blue by UV−vis spectroscopy is then used to determine its concentration and, by extension, the amount of H 2 S that was trapped as ZnS. This method has been used for decades to report the levels of H 2 S due to its simplicity and ease of use. 77,78 We initially investigated adding Zn(II) directly to the buffer with the loaded PLA microparticles, but a white, turbid solution was observed. To address this problem, the Zn(II) salt was added to the methylene blue solution (test tube inside the centrifuge tube), as shown in Figure 9a. When H 2 S was released from the buffer with the loaded PLA microparticles, it partitioned into the atmosphere and then into the centrifuge tube, where it was trapped by the Zn(II) to yield ZnS particles. To minimize oxidation of H 2 S promoted by light, the system was covered in Al foil.
In the experiments reported in Figure 9, 500−2000 μm-sized microparticles loaded with either 0, 9.1, or 16.7% tBDPA were added to water buffered at a pH of 6.7. Each experiment was repeated in triplicate, and the error bars are shown in Figure  9b. The results demonstrate that PLA without tBDPA did not release H 2 S as expected but that as the amount of tBDPA increased in the PLA, the amount of H 2 S released also increased. The microparticles loaded with 9.1% tBDPA had a slower, steadier increase in the amount of H 2 S released than the microparticles loaded with 16.7% tBDPA. The 16.7% loaded phenethylamine hydrochloride in PLA microparticles that lack tBDPA were used as the control. It failed to show any H 2 S releases, which demonstrated that H 2 S was released because of tBDPA in the microparticle.
Growth of Radish Plants Using PLA Microparticles Loaded with 16.7% tBDPA. Chemicals that slowly release hydrogen sulfide have been investigated in agriculture to understand how the release of hydrogen sulfide affects their growth. Dithiophosphates are a new example of these chemicals and have been shown to increase the biomass of corn plants after 4.5 weeks of growth and to increase the harvest yield of corn by 4% after administration of a

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Article dithiophosphate once at planting of the seeds. In studies with dithiophosphates and other chemicals that slowly release H 2 S, chemicals are typically placed on seeds or in soil immediately adjacent to seeds. 44 A challenge of chemicals used to deliver H 2 S is they are typically water-soluble and will diffuse away from plants when watered; this problem may be particularly acute after heavy rainfall. In contrast, PLA microparticles will remain stationary in soil and release dithiophosphates in the same location.
To investigate if PLA loaded with tBDPA could better promote the growth of plants than tBDPA not encapsulated within PLA, the growth of radish plants was investigated. Radish seeds were planted in individual pots and different amounts of free tBDPA, PLA microparticles (500−2000 μm) loaded with tBDPA (16.7% by weight), or PLA microparticles that did not contain tBDPA were added ( Figure 10). The microparticles or free tBDPA were added to the top of the seeds at planting, and 30 seeds were planted for each loading for each additive. For instance, a total of 210 seeds were planted with PLA microparticles loaded with tBDPA, where the loaded PLA was planted with 1, 3, 5, 10, 30, 100, 300, and 600 mg per seed. An additional 210 seeds were planted and exposed to the same loadings of PLA not loaded with tBDPA.
Seeds planted with PLA were investigated to determine the effect of PLA on radish plants since it was known from prior work that PLA can have a small, positive effect on plants. 79 The loaded PLA microparticles were investigated to determine how the slow, localized release of tBDPA impacts the growth of radish plants. The seeds were watered daily with tap water and grown for 4.5 weeks, and then the radishes were harvested and weighed. In each of the application rates as shown in Figure 11, the weight refers to the weight of the PLA

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pubs.acs.org/acsagscitech Article microparticles loaded with tBDPA. For instance, the "1 mg" loading refers to 1 mg of 16.7% tBDPA microparticles. The amount of free tBDPA added at each loading in Figure 10 was the amount of tBDPA within the microparticles at that loading. The results in Figure 11 show that tBDPA loaded in PLA has a significant, positive effect on the growth of radish plants. The weight of the radish plants showed statistically significant improvement in growth when tBDPA loaded into PLA was added at loadings of 10, 30, 100, 300, and 600 mg compared to the control plants not exposed to any chemicals. The weight of the radish plants increased by 141% when only 30 mg of tBDPA-loaded microparticles were used per seed compared with the weight of the plants grown in the absence of these microparticles.
The radishes had the largest increase in weight when grown with PLA microparticles loaded with tBDPA. For all but the 1 mg application rate, radish plants grown in the presence of loaded PLA microparticles had larger weights than radishes grown in the presence of free tBDPA not loaded into PLA or microparticles of PLA without tBDPA. Application of PLA microparticles had a small, positive effect on the growth of the radishes, which is consistent with prior work that demonstrated this effect. It is likely that the degradation of PLA releases carbon into the soil that can improve the growth of radishes. 79 In summary, we developed a method to encapsulate a dithiophosphate salt within PLA at different loadings to control the location and amount of H 2 S released near the roots of radish plants. The dithiophosphate had a rapid release of H 2 S that was complete within 30 days and hydrolyzed to release safe, natural chemicals. PLA is well known to be safe in the environment and is used in numerous agricultural products. By encapsulating a dithiophosphate within PLA, a dual method to control the release of H 2 S was developed. As the PLA hydrolyzes with water, it releases the dithiophosphate which hydrolyzes to release H 2 S. Using the degradation of PLA to slowly release dithiophosphate, the rate at which H 2 S was released to the radish plants differed from the rate of release of H 2 S from dithiophosphates not loaded into PLA. This dual method to control the release of H 2 S had a strong, positive effect on the harvest yield of radishes and points to the importance of adding control over the release of H 2 S using encapsulation. Loading dithiophosphates into PLA not only  . Average radish root weight of plants exposed to various doses of PLA (gray), free tBDPA (orange), and 16.7% tBDPA-loaded PLA (blue) is shown. a = statistical significance to zero control (radish plants without any chemicals) using the Tukey−Kramer test with a 99% confidence interval. A line connecting two groups indicates statistical significance using the Tukey−Kramer test. **99% confidence interval.

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Article provides an extra level of control over the release of H 2 S but also allows the dithiophosphates to be released near the roots of plants. This is important because the dithiophosphates or other chemicals that are commonly used to release H 2 S are water-soluble and can diffuse away in soil particularly when the chemicals are applied early in the growth of plants. We believe that the method reported in this article will advance the investigation of the effect of H 2 S on plants.
■ ASSOCIATED CONTENT

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsagscitech.2c00179. Calibration curve for absorbance of methylene blue, NMR spectra of tBDPA, SEM micrographs of microparticles loaded with tBDPA (PDF)